This invention relates to a process for producing L-proline. More particularly, it relates to a process for the production of L-proline in high yields in the absence of light by cultivation and work-up of a certain type of algae, specifically Chlorella sp. 580. Conditions of cultivation and work-up are defined.
L-proline is a useful amino acid which is used as a medicine and in other applications. In the past, L-proline has been produced mainly by isolation from hydrolysates of proteins or gelatin or by organic synthesis. However, the yield of product obtained by these methods is very low, and the procedures involved are complicated. As a result, L-proline is one of the most expensive amino acids. Therefore, the development of a process for the mass production of L-proline using inexpensive starting materials would be highly desirable.
The fact that L-proline is contained in algae of the genus Chlorella is known. Further, it is known that L-proline is produced by the Chlorella algae in response to the environment in which it is grown i.e. the sodium chloride content of the growth medium. The relationship between L-proline cell accumulation is Chlorella emersonii and sodium chloride content of the environment external to the cell has been described in AUST. J Plant Physiology (1979) 6, 69-79 in which it was reported that in the emersonii species of Chlorella, L-proline production within the cell generally increases as the sodium chloride concentration is increased in the environment external to the cell. However, it appears that only small amounts of L-proline are produced within Chlorella emersonii and, at sodium chloride concentrations higher than 335mM, the cells plasmolyze.
The fact that Chlorella sp. 580 excrete proline also is known as reported in Limnol. Oceanographer (1965) 10:192-206. However, the excretion levels reported therein also are small.
Methods for producing L-proline by fermentation also are known. For example, British 1,132,036 discloses that L-proline can be produced from mutant strains of Micrococcus glutamicus by fermentation. Also U.S. Pat. No. 4,224,409 discloses that L-proline can be obtained by culturing a mutant of the genus Brevibacterium, Corynebacterium or Microbacterium. L-proline production by fermentation, however, is somewhat expensive since the sources of carbon for use in the fermentation medium include carbon sources other than CO.sub.2, such as, for example, pentoses, hexoses, disaccharides, or the like.
Thus, even though it is known that L-proline can be produced both intracellularly and extracellularly by the genus Chlorella, insofar as Applicant is aware, nothing has been reported in the literature with respect to the identification of a specific species of Chlorella and a specific method of cultivating said specific organism such that L-proline can be produced intracellularly within the organism in amounts high enough to be considered sufficient to form the basis for an industrial process.
In Applicant's co-pending U.S. application Ser. No. 329,226, filed Dec. 10, 1981, entitled Process for the Preparation of Amino Acids, Applicant has identified both a specific species of Chlorella, namely Chlorella sp. 580, and a method of cultivating the species to produce L-proline in amounts high enough to be considered sufficient to form the basis for commercial production. According to Applicant's process disclosed therein, Chlorella sp. 580 is cultivated under high-intensity illumination in an aqueous growth medium containing a high concentration of sodium chloride of at least 1M in the final stage of cultivation, in the presence of an adequate supply of carbon, in a depth not exceeding approximately 20 cm of the aqueous medium, until algae of high L-proline content are obtained. The algae are then harvested and L-proline is thereafter recovered from the algae. While Applicant's aforedescribed process does provide for the accumulation of high amounts of L-proline within Chlorella sp. 580 algae cells (up to approximately 35% of the cell weight), cultivation of the algae must be carried out under high-intensity illumination because the process uses as its carbon source, carbon dioxide. That is, fixation of CO.sub.2 by the algae to make cellular material, including L-proline, requires the presence of light. At high cell densities, however, light becomes limiting in that as the cell density of the culture progressively increases during growth, it becomes increasingly more difficult for the light to penetrate the cell mass. This results in a disruption in cell growth and a subsequent decrease in proline production due to the diminished ability of the algae to transform CO.sub.2 into proline due to the lack of light. Thus, it would be highly desirable to supply the cells with a source of carbon which would either supplement or replace CO.sub.2 as a source of proline as the amount of light available to the algae decreases or becomes absent altogether.